Abstract The translation elongation cycle is the central process required for the correct decoding of mRNA into protein. Despite the extremely high level of fidelity that has been observed in this reaction, there are special circumstances that redefine the coding potential of a given mRNA. The incorporation of the 21st amino acid, selenocysteine (Sec), is one example. The transformation of a UGA stop codon into a Sec codon requires the utilization of a novel translation elongation factor (eEFSec), a selenocysteine insertion sequence (SECIS) element in the 3' untranslated region of selenoprotein mRNAs, and a novel SECIS binding protein termed SBP2. These factors act in concert to alter the coding potential of specific UGA codons by specifying the insertion of the Sec-specific tRNA, Sec-tRNASec. This process is required for the production of 25 human selenoproteins, many of which form an essential line of defense against oxidative stress. The proteins are also responsible for a myriad of other functions including thyroid hormone metabolism and maintaining proper sperm motility. We have recently demonstrated that the factors known to be essential for Sec incorporation are, in fact, sufficient. These factors include a specialized elongation factor (eEFSec), a SECIS binding protein (SBP2), a specialized transfer RNA (Sec-tRNASec) and mammalian ribosomes. Although some information about which protein domains are important for Sec incorporation activity, the mechanism by which these factors allow Sec incorporation is not known. A complete understanding of this process is required for three key reasons: 1) many selenoproteins are essential; 2) mutations in the SBP2 gene cause human disease; 3) modulation of selenoprotein production has tremendous potential to maximize the beneficial antioxidant effects of selenoproteins. We propose to address three central questions in this project: What is the molecular basis for the exquisite specificity of Sec incorporation at defined UGA codons? How are cellular dynamics involved in the specificity and efficiency of Sec incorporation? How does the 3' UTR regulate the efficient and processive incorporation of 10 selenocysteine residues into the plasma selenium carrier protein selenoprotein P (SEPP1)? Successful completion of these Aims will shed significant light on the molecular mechanism of Sec incorporation, setting the stage for identifying the means by which the process can be regulated in a clinical setting.